From the analysis of amino acid sequences and secondary and tertiary structures of proteins, a large number of the proteins are composed of separate domains (or modules). For the proteins, a domain is referred to a separate functional and/or structural unit. At least one identical domain may be distributed in various proteins, and one protein may be composed of various different domains. Specific information of the domain may be searched on web sites for bioinformatics, such as Prosite (Hulo N., et al., Nucleic Acids Res, 36:D245-249, 2008; Website: http://kr.expasy.org/prosite/), SMART (Letunic I., et al., Nucleic Acids Res., 34:D257-D260, 2006; Website: http://smart.embl-heidelberg.de/), and representative examples of the domain include immunoglobulin-like, fibronectin II and III, Kringle, etc.
Interactions between biomolecules (for example, protein-protein, protein-nucleic acid interactions) play important roles in various life phenomena such as growth, differentiation and development of cells, intercellular/intracellular signal transductions, and mass transport. As known molecules that specifically bind to target molecules to control the biological activities of the target molecules, antibodies (full-length antibodies or their fragments) have been under leading development. However, the antibodies have various problems in that they are purely expressed and low in solubility, they should be expressed in an animal cell-expressing cell line, the purification costs are very expensive, and their stabilities are very low in the reducing intracellular environment. In order to solve the above problems, there have been attempts to develop proteins, other than the antibodies, that specifically bind to target molecules, such as antibodies, while solving the problems regarding the antibodies (Review Article: Hey, et al., Trends in Biotech. 23:514-522, 2005; Skerra, Current Opin. Biotech., 18:295-304, 2007). These proteins are referred to as protein scaffold, alternative protein scaffold, alternative scaffold, non-antibody protein scaffold, or alternative binding proteins (hereinafter, as referred to as ‘protein scaffold’) (Skerra A, FEBS J, 275:2677-2683, 2008; Skerra, Current Opin. Biotech., 18:295-304, 2007; Nygren P., et al., J. Immunol. Method, 290:3-28, 2004). A model protein scaffold is prepared by constructing a protein library by inducing random or designed mutagenesis at residues or loop structures of a protein exposed from a surface thereof while conserving an amino acid sequence that gives the structural stability in order to maintain its structural scaffold, and separating a variant that specifically binds to a target molecule.
Kringle domains occur as separate modules in proteins of various species including human, and several tens or hundreds Kringle domains are present in one protein (see Table 1) (Castellino, et al., J Mol Evol, 26:358-369, 1987; Ikeo, et al., J Mol Evol, 40:331-336, 1995; Cao, et al., Curr Med Chem-Anti-Cancer agents, 2:667-681, 2002). For a variety of living organisms existing in the nature, 1663 different kinds of Kringle domains have been founded from 893 proteins, and 39 Kringle domains whose amino acid sequences are different from each other are distributed in 31 human proteins (See: Prosite (Hulo N., et al., Nucleic Acids Res, 36:D245-249, 2008; Website: http://kr.expasy.org/prosite/; SMART (Letunic I., et al., Nucleic Acids Res, 34:D257-D260, 2006; Website: http://smart.embl-heidelberg.de/) (Castellino, et al., J Mol Evol, 26:358-369, 1987; Ikeo, et al., J Mol Evol, 40:331-336, 1995). A loop (inter-Kringle domain) including approximately 20 amino acids is present between the Kringle domains. The exact functions of the Kringle domains are not known, but the Kringle domains play a role in binding to various biomolecules (for example, proteins, peptides, carbohydrates, cell membranes, phospholipids and the like) to give the binding activity to corresponding proteins and controlling various biological activities of the corresponding proteins (Cao, et al., Curr Med Chem-Anti-Cancer agents, 2:667-681, 2002). Kringle domains are typically distributed in growth factors, proteases, blood coagulation factors, transmembrane receptors, and the like (Castellino, et al., J Mol Evol, 26:358-369, 1987). In particular, the Kringle domains are present independently from the other proteins, or present with the other proteins (for example, endostatin, angiostatin), and also serve to inhibit angiogenesis (antiangiogenesis). Angiostatin has 4 Kringle domains, and this structure of the angiostatin is necessarily used to inhibit angiogenesis (Cao, et al., Curr Med Chem-Anti-Cancer agents, 2:667-681, 2002). Lysine binding sites of Kringle domains in plasminogen and plasmin, which are associated with the fibrinolysis, have been known to be binding sites between extracellular matrix molecules of the two proteins and cell receptors, and thus the lysine binding sites of the Kringle domain has been considered to be important for the fibrinolysis that is the function of the two proteins (Cao, et al., Curr Med Chem-Anti-Cancer agents, 2:667-681, 2002).
TABLE 1No. ofKringleProtein namesdomainsProthrombin2Plasminogen5Urokinase-type plasminogen activator (uPa)1Tissue-type plasminogen activator (tPa)2Blood coagulation factor XII (Hagenman factor)1Apolipoprotein A38Hepatocyte growth factor/Scatter Factor (HGF/SF)4Macrophage-stimulating protein (MSP)/HGF4like proteinHGF activator1Kremen1Neurotrypsin/Motopsin1Plasma hyaluronan binding protein (PHBP)1Serine protease (Hermandid momus)1ROR 1&21Drosophila neurospecific receptor kinase1Drosophila receptor kinase1C. elegans ROR receptor tyrosin kinase1Muscle specific tyrosin kinase (Musk) (Torpedo,1Xenopus)
Proteins including Kringle domains, and the number of Kringle domains in the proteins are listed in Table 1.
Kringle is a domain or module that has an independent tertiary structure at a variety of proteins in various living organisms, such as humans. A typical Kringle domain is composed of approximately 80 amino acids, and has a tertiary structure connected with 3 disulfide bonds (S—S bond), which provides structurally strong loop structures (Castellino, et al., J Mol Evol, 26:358-369, 1987; Ikeo, et al., J Mol Evol, 40:331-336, 1995; Castellino, et al., Ciba Found Symp, 212:46-60, 1997; Marti, et al., Biochemistry, 38:15741-15755, 1999). The typical Kringle domain has three 1-6, 2-4 and 3-5 disulfide bonding patterns. That is, the disulfide bonding patterns are formed between cysteines 1 and 80, between cysteines 22 and 63, and between cysteines 51 and 75 (Ikeo, et al., J Mol Evol, 40:331-336, 1995; Castellino, et al., Ciba Found Symp, 212:46-60, 1997; Marti, et al., Biochemistry, 38:15741-15755, 1999).
Kringle domains have been found in more than 893 proteins (at least 31 human proteins) from the living organisms living in the nature. In the case of the Kringle domains, some of their amino acids that give the structural stability were conserved, but the other amino acids were not conserved at an amino acid or nucleotide level. Accordingly, it is necessary to construct a variant library by conserving an amino acid sequence of the Kringle domain, which gives the typical structural characteristics, to form a structural scaffold (for example, 3 intracellular disulfide bonds (with 1-6, 2-4 and 3-5 disulfide bonding patterns) and thus a prepared loop structure) and introducing designed or random mutations into structurally flexible regions of the loop structures to have various combinations of amino acid sequences that do not exist in the nature, and also to characterize the variants based on the Kringle domain structural scaffold, which specifically bind to a variety of target molecules from the designed library, by separating and identifying the variants. Also, since the Kringle domain structure-based variants control the biological activities of the target molecules, they may be used to develop methods and compositions for prevention, detection, diagnosis, treatment and relieving of various diseases.